Increasing the Sensitivity of Enzyme-Linked
Immunosorbent Assay Using Multiplexed Electrokinetic
Concentrator
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Citation
Cheow, Lih Feng et al. “Increasing the Sensitivity of EnzymeLinked Immunosorbent Assay Using Multiplexed Electrokinetic
Concentrator.” Analytical Chemistry 82.8 (2010): 3383–3388.
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http://dx.doi.org/10.1021/ac9024335
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American Chemical Society (ACS)
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Wed May 25 21:52:56 EDT 2016
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http://hdl.handle.net/1721.1/73020
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Author Manuscript
Anal Chem. Author manuscript; available in PMC 2011 April 15.
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Published in final edited form as:
Anal Chem. 2010 April 15; 82(8): 3383–3388. doi:10.1021/ac9024335.
Increasing the Sensitivity of ELISA using Multiplexed
Electrokinetic Concentrator
Lih Feng Cheow1, Sung Hee Ko2, Sung Jae Kim1, Kwan Hyoung Kang2, and Jongyoon
Han*,1,3
1Department of Electrical Engineering and Computer Science, Massachusetts Institute of
Technology, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139
2Department
of Mechanical Engineering, San 31, Pohang University of Science and Technology,
Hyoja-dong, Pohang, Korea 790-784
3Department
of Biological Engineering, Massachusetts Institute of Technology, 77 Massachusetts
Avenue, Cambridge, Massachusetts 02139
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Abstract
We developed a novel method to increase the sensitivity of standard Enzyme-Linked Immunosorbent
Assay (ELISA) using a multiplexed electrokinetic concentration chip. The poly(dimethylsiloxane)
(PDMS) molecular concentrator1 was used to trap and collect charged fluorescent product of targetbound enzyme turnover reaction of ELISA that occurred in a standard 96 well plate. Detection
sensitivities of both Prostate Specific Antigen (PSA) and CA 19-9 (a human pancreatic and
gastrointestinal cancer marker) ELISAs in serum are enhanced ~100 fold with a low CV of <17%.
We also integrated this method with an on-chip bead-based ELISA that lends itself toward a fullyautomated on-chip diagnostic device. Detection sensitivity of microfluidic bead-based CA 19-9
ELISA in serum is enhanced ~65 fold compared to the results without electrokinetic accumulation
step. This chip can be directly applied to enhance the readout sensitivity of a wide range of existing
ELISA kits at concentrations below the current detection limit.
INTRODUCTION
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One of the most pressing issues in analytical chemistry is detecting low abundance analytes
from complex samples such as blood. Tests for low abundance biomarkers such as human
prostate specific antigen (PSA) and human pancreatic and gastrointestinal cancer marker (CA
19-9) are routinely used for initial tumor screening, to monitor patient response during cancer
therapy, and to help detect recurrence following treatment2, 3. Therefore, there is significant
clinical relevance to increase the sensitivity of these tests to facilitate early detection of the
onset and recurrence of cancer, since these biomarkers get diluted significantly in blood,
especially in early stages of the disease progression. Among various new and old immunoassay
formats, ELISAs (Enzyme-Linked Immunosorbent Assays) are probably the most widely-used
techniques, due to their simplicity, flexibility and the enzymatic signal amplification enabling
high sensitivity4. Still, the ultimate detection sensitivity of ELISA is not good enough to tackle
the challenges of biomarker detection. This led to the development of many novel ultrasensitive
immunoassay platforms5-8. However, additional sensitivity comes with the cost of added
complexity in amplification chemistry or complicated readout devices. Being able to measure
analytes at concentrations below the current detection limits of ELISA, without modifying the
* To whom correspondence should be addressed. jyhan@mit.edu Phone: +1-617-253-2290. Fax: +1-617-258-5846 .
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basic workflow of ELISA (therefore applicable to any existing ELISA setup), would be very
useful.
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Our group has developed nanofluidic preconcentration devices that can be used to continuously
collect and trap charged molecules in a given sample into a much smaller volume, thereby
increasing local concentration significantly1, 9. These devices have been used to enhance
surface-bound protein binding kinetics10 as well as to increase the sensitivity of homogeneous
enzyme assay11, 12. While these methods demonstrated superior sensitivity, they were tailored
for the specific molecules and need to be optimized for concentrating other molecules.
Furthermore, these devices were not demonstrated in a multiplexed format.
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In this paper, we utilize a multiplexed nanofluidic electrokinetic concentrator to accumulate
fluorescent product molecules from ELISA in a small volume. Instead of aiming to affect the
primary immunobinding by concentrating binding partners10, here we simply aim to collect
the fluorescent product of standard ELISA complex, without modifying the immunobinding
process of ELISA. By increasing the local concentration of reporter molecule by several orders
of magnitude at the detection zone, we achieved ~65-100 fold enhancements in detection limit
of two cancer markers. We also demonstrated multiplexing capability by performing
concentration-enhanced ELISA of cancer biomarkers from five different samples concurrently,
thus enabling us to obtain a dose response curve in one single experiment. Since the same
substrate can be used for a whole range of ELISA kits, the same device can be directly applied
to immunoassay of different analytes. As a proof of concept, we performed our experiment
with two cancer markers, PSA and CA 19-9, which are two important biomarkers in cancer
diagnostic and post treatment monitoring2, 3.
I. CONCENTRATION ENHANCED MICROWELL ELISA
EXPERIMENTAL SECTION
Microchip Fabrication—The microchip was fabricated using poly(dimethysiloxane)
PDMS (Sylgard 184, Dow Corning Inc., Midland, MI) irreversibly bonded on a glass slide.
Microchannels were molded in PDMS by replica molding technique13. To obtain the positive
master mold, the desired design was photolithographically patterned onto a silicon wafer using
positive photoresist. Next, the wafer was etched to a depth of 15 μm via a reactive ion etching
(RIE) process. The silicon master was further treated with trichlorosilane (T2492, UCT
Specialties, Bristol, PA) in a vacuum desiccator overnight to prevent adhesion to PDMS.
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We fabricated the ion-selective nanoporous structures by using the microflow patterning
technique to obtain a thin strip of Nafion film on a standard glass slide1, 11. A 50 μm deep and
400 μm wide PDMS microchannel was used to define the flow path of the Nafion solution (20
wt% solution in lower aliphatic alcohol/H2O mix, Sigma Aldrich, St. Louis, MO). The PDMS
chip with microchannels was irreversibly bonded on top of the glass slide by standard plasma
bonding.
Materials—We tested our microfluidic chip on two commercially available ELISA kits for
common biomarkers. The first was a human prostate serum antigen (PSA) ELISA kit (Catalog
#8425-300, Monobind Inc., Lake Forest, CA). The second kit was a human pancreatic and
gastrointestinal cancer marker (CA 19-9) ELISA kit (Catalog #1840, Alpha Diagnostics
International Inc., San Antonio, TX).
We used Amplex Red (A12222, Invitrogen Corp., Carlsbad, CA) as the fluorogenic enzyme
substrate. This colorless substrate reacts with hydrogen peroxide with a 1:1 stoichiometry to
produce highly fluorescent resorufin14 (excitation/emission maxima = 570/585 nm). FITC dye
(F143, Invitrogen Corp., Carlsbad, CA) was used as a tracer to monitor electrokinetic
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accumulation under a different filter set (excitation/emission maxima = 490/513 nm). Donkey
serum was obtained from Innovative Research, Novi, MI. Electrokinetic product accumulation
was done in 0.01X PBS with pH 7.4 as the buffer solution.
Sample Preparation—Immunobinding reaction of PSA and CA 19-9 was performed in
donkey serum and control serum prepared from human sera respectively. Twenty five μL of
serially diluted antigen sample in serum was added to a 100 μL mixture of biotinylated primary
antibody and HRP-coupled secondary antibody in a Streptavidin coated microwell strip. After
60 minutes incubation at room temperature, the microwell contents were discarded by
decantation and washed six times with wash buffer. Finally, 100 μL of freshly prepared
substrate solution (containing 20 μM Amplex Red, 10 μM H2O2, and 100 μM FITC tracer in
0.01X PBS buffer) was added to all wells. After 30 minutes incubation, the contents of the
wells were collected in separate microcentrifuge tubes and used immediately or stored at -20
°C and thawed to room temperature before using.
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Device Scheme and Operation—A schematic of the multiplexed PDMS concentrator is
shown in Figure 1. The chip contained five sample loading inlets connected to one common
outlet. See Supplementary Material for the fabricated device. Two side channels flanked the
inlet channels to provide symmetrical electrical ground. An ion-selective nanojunction was
fabricated at the center of the device to concentrate the fluorescent product molecules by
electrokinetic trapping when voltages were applied. We have also fabricated arrays of pillar
structures in front of the nanojunction, which helped to stabilize the location of the
electrokinetically trapped molecules by reducing vortexes formed in the ion depletion zone
during concentration polarization15.
Figure 2a showed a detailed operation of the device. Conventional ELISA was performed
entirely in microwell strips as described in the sample preparation section. Next, reaction
products corresponding to different antigen concentrations were loaded into each inlet
channels.
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To electrokinetically trap the charged fluorescent enzymatic product molecules, we applied 30
V to all the inlet and outlet reservoirs for 10 minutes, while grounding the side channels. An
ion depletion region was induced near the ion-selective Nafion membrane by concentration
polarization effect16, 17. Meanwhile, liquid height difference between the inlet and outlet
reservoirs (15 μL sample in inlet reservoirs, 10 μL buffer in outlet reservoir) induced a
gravitational flow that continuously stacked the charged enzyme product molecules against
the ion depletion zone. We performed this experiment three times in the same device to
investigate intraassay repeatability. Between each experiment, the channels were flushed with
deionized water for 5 minutes to remove carryover from the previous experiment.
Measurement Instrument and Image Analysis—An inverted epifluorescence
microscope IX 71 (Olympus, Center Valley, PA) equipped with a cooled CCD camera
(SensiCam, Cooke Corp., Romulus, MI) was used for fluorescence imaging. A mechanical
shutter (Olympus, Center Valley, PA), which only opens for 100 ms when images are taken,
was used to prevent photobleaching of the fluorescent molecules. It had been reported that
strong excitation light led to enzyme independent autocatalyzed photooxidation of Amplex
Red to fluorescent resorufin18. We took images every 5 s and engaged two neutral density
filters (ND6) during imaging to reduce this effect. The images were analyzed using the NIH
ImageJ software. Nonuniformity in background signal was corrected by subtracting an image
of the device filled with deionized water taken before each experiment. To quantify the
concentrated product molecules, we plotted a profile of the channel-width-averaged
fluorescence intensity and picked the maximum value. To quantify the product molecules
without electrokinetic accumulation, we measured the channel-width-averaged fluorescence
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intensity in a region far before the concentrated plug. Origin7 software (OriginLab Corp.,
Northampton, MA) was used to fit the dose response curve.
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RESULTS AND DISCUSSION
Mechanism of Multiplexed Electrokinetic Accumulation—The overall design of
multiplexed electrokinetic accumulation was different from previously reported, single-gate
or double-gate devices1, 9-12. In the multiplexed devices shown in this paper, not all the sample
channels were directly adjacent to a grounded side channel, yet electrokinetic accumulation
still occurred in each channel, with approximately the same efficiency (checked by monitoring
FITC accumulation). We believe that there were alternative paths through the ion selective
membrane directly connecting each sample channel to the side channels. In the microflow
patterning method1, 11, the 400 μm wide planar Nafion membrane was found to be ~ 1 μm
thick by surface profiling. Cations from the inner sample channels could travel within the
Nafion matrix to the outermost side channels. Further experiments are being conducted now
to verify this hypothesis.
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Multiplexed Concentration Enhanced ELISA—We spiked donkey serum with known
amounts of PSA (1000 pg/mL, 200 pg/mL, 40 pg/mL, 8 pg/mL, 1.6 pg/mL) to simulate the
effect of a complex sample matrix. Conventional ELISA of these samples was performed in
microwell strips, and the enzymatic reaction was allowed to proceed for 30 minutes. After this,
the enzymatic products were injected into the microchip for electrokinetic accumulation.
Figure 2b showed uniform accumulation of the tracer molecule, which indicated similar
accumulation rate. Tracer molecules at the top channel accumulated at a different location from
the rest of the channels because the high concentration of charged product molecules modified
the ionic environment at the vicinity of the nanojunction19 (Figure 2b). Figure 2c showed
electrokinetic accumulation of enzymatic product molecules of PSA ELISA. Without
electrokinetic accumulation, only the top channel, which corresponded to high PSA
concentration (1000 pg/mL), showed visible product fluorescence. However, the fluorescence
intensity in all five channels was markedly increased in the accumulation zone. Figure 2d
showed the dose response curve of PSA ELISA in both normal mode and accumulation mode.
The error bars represented one standard deviation for three separate experiments. As it was
unclear the effect of electrokinetic accumulation on the dose response curve, we fitted it to a
second order polynomial equation. Zero-dose response was obtained by simultaneous
accumulation of zero-dose ELISA products in three channels. The limit of detection (LOD)
corresponds to the analyte concentration needed to produce a signal three standard deviations
above the zero dose response. Using this criterion, the LOD of PSA ELISA with electrokinetic
accumulation was 1.85pg/mL, compared to 193.5pg/mL without electrokinetic accumulation,
representing ~100-fold sensitivity enhancement.
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To show that this method is general and can be applied to multiple analyte, we performed a
similar experiment using CA 19-9 as the target antigen. We spiked human control serum with
known amount of CA 19-9 (6.25 U/mL, 1.25 U/mL, 0.25 U/mL, 0.05 U/mL and 0 U/mL) and
repeated the above experiment. The result (SI Figure 2 in Supplementary Material) showed the
dose response curve of CA 19-9 ELISA in both normal mode and accumulation mode. The
LOD of CA 19-9 ELISA with electrokinetic accumulation was 0.011 U/mL, compared to 1.2
U/mL without electrokinetic accumulation, representing ~110-fold sensitivity enhancement.
II. CONCENTRATION ENHANCED ON-CHIP BEAD BASED ELISA
Realization of point-of-care (POC) testing is difficult with conventional immunoassay, since
rather large devices are necessary for automated practical diagnosis systems20. A fully
automated portable micro-ELISA system is recently demonstrated as a viable POC tool 21. In
micro-ELISA, immunobinding reaction occurs at the surface of polystyrene beads that are
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captured within the microchannel. Dye molecules produced by enzymatic reaction upon
introduction of substrates are detected downstream of the bead capture zone. However, microELISA suffers from poor sensitivity in continuous-flow condition due to the short enzymatic
reaction time21. Higher sensitivity could be achieved in the stop-flow mode21, but even in this
case the signal intensity levels off due to equilibrium of dye molecule production and diffusion.
We can provide a simple solution to this problem by accumulating the dye molecules
downstream of the reaction zone using an electrokinetic concentrator. This would make microELISA a powerful ultrasensitive POC diagnostic tool, applicable to a wide range of low
abundance targets. As a proof of concept, we performed concentration enhanced bead-based
ELISA of the cancer marker CA 19-9.
EXPERIMENTAL SECTION
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Sample Preparation—Twenty fives μL of serially diluted antigen sample in control serum,
50 μL of the biotinylated primary antibody, and 5 μL of Streptavidin coated beads (Applied
Biosystems, Foster City, CA) were added into a microcentrifuge tube and mixed by vortexing.
After 60 minutes incubation at room temperature, the tubes were spun in a centrifuge for 5
minutes at 6000 rpm to obtain a firm pellet of beads. The supernatant was discarded and the
pellet was resuspended in 100 μL of wash buffer. This step was repeated five times to ensure
proper washing. Next, 50 μL of the HRP-coupled secondary antibody solution was added into
each tube and incubated for 60 minutes at room temperature, followed by six washing steps as
described above. Finally, the beads were resuspended in 100 μL 0.01X PBS buffer.
Device Scheme and Operation—Figure 3a showed a detailed procedure and the operation
of the device in each inlet channel. Beads from different samples were loaded into each inlet
reservoirs, and a negative pressure was applied at the outlet reservoir to pull in the bead solution.
Since the diameter of the beads (6 – 8 μm) was larger than the gaps between the pillar structures
(5 μm), the beads were trapped in front of the pillar structures. Negative pressure was removed
when the length of the bead pack reached ~500 μm (±15%). Following this, the bead solution
in the inlet reservoir was removed and flushed with deionized water for 30 seconds.
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In the second step, freshly prepared substrate solution (containing 20 μM Amplex Red, 10
μM H2O2, and 100 μM FITC tracer in 0.01X PBS buffer) was injected into the inlet reservoirs.
Electrokinetic trapping of charged fluorescent product molecules was similar to the previous
experiment, with a 20 V potential difference between the inlet channels and side channels, and
gravitational flow due to 40 μL sample in the inlet reservoirs vs. 20 μL buffer in the outlet
reservoir. We performed this experiment three times in the same device using the same
parameters to investigate intraassay repeatability. Between each experiment, the channels were
flushed with deionized water for 5 minutes to remove carryover from the previous experiment.
RESULTS AND DISCUSSION
Multiplexed Concentration Enhanced Bead-Based ELISA—Figure 3 showed
simultaneous accumulation of ELISA products for beads incubated with serial dilution (6.25
U/mL, 1.25 U/mL, 0.25 U/mL, 0.05 U/mL and 0 U/mL) of CA 19-9 in control serum. Uniform
bead packing was achieved in all five channels. From Figure 3b, only the top channel which
contains beads with high surface concentration of antigen appeared slightly fluorescent
immediately after the beads, indicating the low assay sensitivity. However, at the vicinity of
the nanojunction, electrokinetic trapping led to accumulation of the charged fluorescent
product molecules and clear increase in signal intensity in all five channels. Figure 3c showed
the dose response curve of CA 19-9 ELISA in both normal mode and accumulation mode,
fitted to a second order polynomial equation. The LOD of CA 19-9 ELISA with electrokinetic
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accumulation was 0.02 U/mL, compared to 0.13 U/mL without electrokinetic accumulation,
representing approximately 65 fold sensitivity enhancement.
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Repeatability Studies—Repeatability of immunoassay is an important figure of merit, since
it is directly related to the issues of false-positive and false-negative biomarker detection, both
with serious clinical consequences. We characterized the intra-assay reproducibility of the
multiplexed concentration enhanced ELISA by measuring the coefficient of variation (CV).
We calculated the CV as the standard deviation σ divided by the mean fluorescence μ of three
separate experiments for biomarker concentration (CV = σ/μ). As shown in Table 1, the CV
of microwell PSA and CA 19-9 ELISA with electrokinetic accumulation was below 17% across
the entire dynamic range, showing good reproducibility of our assay. In comparison, the
maximum CV for bead based CA 19-9 ELISA was 37%. We attribute the increase in CV to
the variation of the number of beads (±15%) in each channel, which leads to different
hydrodynamic resistance and enzymatic reaction time. It had been reported that the substrate
turnover efficiency in an enzymatic packed bead reactor is a function of flow rate22. Hence,
variations in bead packing could lead to errors in microfluidic bead based ELISA. Secondly,
due to variable zeta potential in the differently functionalized beads in each channel, different
electroosmotic flow was induced in each channel. Furthermore, beads could be dislodged from
the bead trap due to backpressure generated during electrokinetic accumulation of charged
molecules.
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Comparison with ELISA on a Plate Reader—Finally, we compared our results with
conventional fluorogenic readout on a 96 well plate reader (Varioskan Flash, Thermo
Scientific, Waltham, MA). Table 2 shows the comparison among the ELISA LOD’s obtained
in the electrokinetic accumulation region, in the microchannel without electrokinetic
accumulation, and in a 96 well plate. Electrokinetic accumulation improves the PSA/CA 19-9
ELISA detection sensitivity by ~100X/110X compared to fluorogenic detection in
microchannel without electrokinetic accumulation, and ~30X/10X compared to plate reader.
The microchannel detection is least sensitive due to its short optical path length (12 μm)
compared to that of 96 well plate (~2mm).
The detection limit of CA 19-9 bead based ELISA in a microchannel without electrokinetic
accumulation (0.13 U/mL) was slightly better to that obtained in a 96 well plate (0.115 U/mL).
This is attributed to the high enzymatic conversion efficiency at the bead pack and confinement
of the fluorescent product to the downstream of the microchannel23. With electrokinetic
accumulation, the detection sensitivity was ~70X better than both cases.
CONCLUSION
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In this paper, we reported a novel multiplexed PDMS microfluidic platform to improve the
readout sensitivity of fluorogenic ELISA. An ion-selective membrane patterned across the
microchannels enabled electrokinetic accumulation of charged fluorescent ELISA products,
thereby increasing local signal intensity in the trapping zone. Up to five assays could be
performed simultaneously in a multiplexed format, enabling assay parallelization for highthroughput immunoassay.
The post-ELISA accumulation step presented here could find wide applications for low
abundant biomolecule detection such as disease marker, cytokines, hormones, and specific
analytes in biological research. In this method, the enzyme substrate that is used here can be
applied to all other HRP-based ELISA without any modification. Hence there is no need to redevelop the assay for a new analyte. The method presented here could capitalize on the
tremendous amount of validation and quality control performed by ELISA kit manufacturers
to produce results with high reproducibility while extending the dynamic range of
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immunoassay towards a lower limit. Due to the simplicity of fabrication and small sample
volume requirement, this microchip is amenable to direct integration with lab-on-chip devices
to provide automation and new capabilities such as single-cell immunoassay.
Supplementary Material
Refer to Web version on PubMed Central for supplementary material.
Acknowledgments
This work was supported by NIH (CA119402 and EB005743). L.F. Cheow is supported by the A*STAR National
Science Scholarship. S.H. Ko is supported by the Korea Science and Engineering Foundation grant
(R0A-2007-000-20098-0). Microfabrication of the device was done in the Microsystems Technology Laboratories of
MIT, with the help of its staff members.
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Figure 1.
(a) Schematic of the multiplexed electrokinetic accumulator, which consists of a Nafion
nanojunction that spans the five inlet microchannels and two side channels. Sandwich ELISA
was performed on 6-8 μm Streptavidin beads, which were then loaded sequentially into the
inlet reservoirs. (b) Microscope image of the fabricated PDMS microchannels integrated with
a surface patterned nanojunction.
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Figure 2.
Concentration-enhanced PSA ELISA. (a) ELISA product from 96-well plates was directly
injected into the microchannels. When a voltage was applied between the inlet microchannels
and the side channels, the product molecules were electrokinetically accumulated and attained
a high local concentration. The sensitivity of fluorescence detection was significantly enhanced
in this region. (b) The concentrated FITC tracer showed similar intensities, indicating similar
accumulation rate in all channels. (c) Without electrokinetic accumulation, only the top channel
(corresponding to high concentration of PSA) displayed a slight increase in product
fluorescence. In the accumulation region, it was easy to differentiate the difference in product
fluorescence between each channel. (d) Dose response curve of concentration enhanced PSA
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ELISA. Square markers represent intensities with product accumulation, while cross markers
represent intensities without product accumulation. Error bars indicate one standard deviation
from the mean of three separate experiments. Sensitivity enhancement due to product
accumulation was ~ 100 fold.
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Figure 3.
Concentration-enhanced bead based CA 19-9 ELISA. (a) Step 1: Functionalized beads were
drawn into the inlet microchannels by applying a negative pressure at the outlet reservoir. The
6-8 μm beads were trapped in front of the 5 μm microfabricated constrictions. Step 2: Substrate
molecules were injected into the inlet microchannels, and were enzymatically converted into
charged fluorescent product molecules in the bead pack. When a voltage was applied between
the inlet microchannels and the side channels, the product molecules were electrokinetically
accumulated and attained a high local concentration. The sensitivity of fluorescence detection
was significantly enhanced in this region. (b) Without electrokinetic accumulation, only the
top channel which contained beads with high surface concentration of antigen displayed visible
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increase in product fluorescence. In the accumulation region, it was easy to differentiate the
difference in product fluorescence between each channel. (c) Dose response curve of
concentration enhanced bead based CA 19-9 ELISA. Square markers represent intensities with
product accumulation, while cross markers represent intensities without product accumulation.
Sensitivity enhancement due to product accumulation was ~ 65 fold.
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6.25
6.2
CA 19-9 Concentration (U/mL)
CV (%)
(Concentration Enhanced ELISA)
23.7
14.6
CV (%)
(Concentration Enhanced ELISA)
CV (%)
(Bead Based
Concentration Enhanced ELISA)
1000
PSA Concentration (pg/mL)
18.7
2.0
1.25
11.4
200
37.2
1.7
0.25
9.9
40
33.6
16.5
0.05
12.7
8
7.5
8.4
0
14.6
1.6
Coefficients of variation for PSA and CA 19-9 ELISA in accumulation mode.
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Table 1
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Table 2
Comparing the limit of detection of PSA and CA 19-9 between different methods.
NIH-PA Author Manuscript
Limit of Detection
(LOD)
Microchannel
(Electrokinetic
Concentration)
Microchannel
(No Electrokinetic
Concentration)
96 well
Plate
PSA in Donkey Serum
(Concentration Enhanced ELISA)
1.85 pg/mL
193.5 pg/mL
64 pg/mL
CA 19-9 in Control Serum
(Concentration Enhanced ELISA)
0.011 U/mL
1.2 U/mL
0.115 U/mL
CA 19-9 in Control Serum
(Bead Based
Concentration Enhanced ELISA)
0.002 U/mL
0.13 U/mL
0.115 U/mL
NIH-PA Author Manuscript
NIH-PA Author Manuscript
Anal Chem. Author manuscript; available in PMC 2011 April 15.